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Abstract:

The present disclosure relates to bisphenyl compounds that are useful for
inhibiting the ADP-ribosyl cyclase (ADPR-cyclase). More particularly, the
disclosed compounds can be used for treatment and prevention of
hypertension, hypertensive cardiac hypertrophy, diabetes, and diabetic
nephropathy, in which pathogenesis ADPR-cyclase is involved. The
compounds and compositions of the invention can be used for treatment and
prevention of cardiovascular disease and related disease states,
particularly, hypertension or diabetes related disorders, such as,
hypertensive cardiac hypertrophy, diabetic nephropathy, and the like.

Claims:

1-9. (canceled)

10. A method of treating or preventing a cardiovascular or renal disease
comprising administering a pharmaceutically acceptable composition
comprising a therapeutically effective amount of a compound of the
Formula I, ##STR00007## wherein: X and Y are selected from C and N;
R1, R2, R3, R4, R5, R6, R7, R8,
R9, and R10 are each independently selected from hydrogen and
hydroxyl; and the bond between X and Y is selected from a single bond or
a double bond.

12. The method of claim 11, wherein the pharmaceutically acceptable
composition comprises the compound of Formula I in a concentration from
about 0.0005 mM to about 50 mM.

13. The method of claim 10, wherein the compound of Formula I is
2,2'-dihydroxyazobenzene for inhibiting heart specific ADPR-cyclase.

14. The method of claim 13, wherein the pharmaceutically acceptable
composition comprises the compound of Formula I in a concentration from
about 0.05 mM to about 100 mM.

15. The method of claim 10, wherein the compound of Formula I is
resveratrol for inhibiting kidney ADPR-cyclase.

16. The method of claim 15, wherein the pharmaceutically acceptable
composition comprises the compound of Formula I in a concentration from
about 0.01 mM to about 100 mM.

Description:

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application is a divisional of Ser. No. 12/520,391,
filed Oct. 27, 2009, which was a national stage application of PCT
Application No. PCT/KR07/06921, filed Dec. 28, 2007, which claims
priority to Korean Application No. 10-2006-0135890, filed Dec. 28, 2006,
the entire disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to bisphenyl compounds that are
useful for inhibit the ADP-ribosyl cyclase (ADPR-cyclase). More
particularly, the disclosed compounds can be used for treatment and
prevention of hypertension, hypertensive cardiac hypertrophy, diabetes,
and diabetic nephropathy, in which pathogenesis ADPR-cyclase is involved.

[0005] Angiotensin II (Ang II) plays a key role in the regulation of
cardiovascular homeostasis. Acting on both the "content" and the
"container" Ang II regulates blood volume and vascular resistance. The
wide spectrum of Ang II target tissues includes the adrenals, kidney,
brain, pituitary gland, vascular smooth muscle, and the sympathetic
nervous system. Angiotensin is not only a blood-borne hormone that is
produced and acts in the circulation but is also formed in many tissues
such as brain, kidney, heart, and blood vessels [Gasparo et al.,
Pharmacol. Rev. 2000(52):415-72]. Recent studies report that Ang II
induces ADPR-cyclase activation and production of cADPR [Fellner et al.,
Am. J. Physiol Renal Physiol. 2005(288): F785-91; Higashida et al.,
Biochem. J. (2000)352:197-202].

[0007] A study with mice disrupted CD38 gene has demonstrated that
formation of cADPR is not reduced greatly in mouse kidney, brain, and
heart [Partida-Sanchez et al., Nat. Med. 2001(7): 1209-16], suggesting
that ADPR-cyclases other than CD38 exist. However, the ADPR-cyclase(s)
present in these tissues has not been cloned, and the cADPR antagonistic
derivatives such as 8-Br-cADPR and 8-NH2-cADPR do not distinguish
the Ca2+ signals induced by the unidentified ADPR-cyclase or CD38
[Walseth et al., Biochim. Biophys. Acta. (1993)1178:235-42].

[0008] The present invention provides available bisphenyl derivatives that
inhibit specific ADPR-cyclase activity with high potency, but not CD38.
These compounds that are thought to contribute to the prevention or
treatment of various diseases, including hypertension and diabetic
nephropathy.

DISCLOSURE OF INVENTION

Technical Problem

[0009] An object of the present invention is to provide novel ADPR-cyclase
inhibitors, which are bisphenyl compounds. Theses molecules exhibit
highly selective and specific inhibition effects in ADPR-cyclase
activation.

[0010] This invention also provides that a small molecule ADPR-cyclase
inhibitor can develop as therapeutic agents for the treatment and
prevention of cardiovascular and renal disease, particularly,
hypertension or hypertensive cardiac hypertrophy and diabetic or
hypertensive nephropathy. Technical Solution.

[0018] The term "pharmaceutically acceptable composition" as used herein
refers to a composition comprising at least one compound as disclosed
herein formulated together with one or more pharmaceutically acceptable
carriers.

[0019] The term "pharmaceutically acceptable salt(s)" refers to salts of
acidic or basic groups that may be present in compounds used in the
present compositions. Compounds included in the present compositions that
are basic in nature are capable of forming a wide variety of slats with
various inorganic and organic acids. The acids that may be used to
prepare pharmaceutically acceptable acid addition salts of such basic
compounds are those that form non-toxic acid addition salts, i.e., slats
containing pharmacologically acceptable anions.

[0020] Substituent around a carbon-carbon double bond or nitrogen-nitrogen
double bond alternatively can be referred to as "cis" or "trans" where
"cis" represents substituent on the same side of the double bond and
"trans" represents substituent on opposite sides of the double bond.

[0021] The present disclosure also provides pharmaceutical compositions
comprising compounds as disclosed herein formulated together with one or
more pharmaceutically acceptable carriers. These formulations include
those suitable for oral, rectal, topical, buccal, and parenteral, such as
subcutaneous, intramuscular, intradermal, or intravenous, administration,
although the most suitable form of administration in any given case will
depend on the degree and severity of the condition being treated and on
the nature of the particular compound being used.

[0022] The amount of active compound administered may be dependent on the
subject being treated, the subject's weight, and the manner of
administration and the judgment of prescribing physician. For example, a
dosing schedule may involve the daily or semi-daily administration of the
encapsulated compound at a perceived dosage of about 0.05 mg to about 35
mg, particularly, about 0.2 mg to about 25 mg.

[0023] The therapeutically effective amount of the compound of Formula I
is sufficient to establish a concentration ranging from about 0.001 mM to
about 100 mM, particularly, from about 0.1 mM to about 20 mM in mammals.

[0024] A therapeutically effective amount of a compound or composition
disclosed herein can be measured by the therapeutic effectiveness of the
compound. Compounds of the invention may be administered in a dose of
about 1 mg/kg to about 50 mg/kg daily. However, the dosages may be varied
depending upon the requirements of the patients, the severity of the
condition being treated, and the compound being used.

[0025] In certain embodiments, the compounds of the invention are useful
for treatment of diseases characterized by activated ADPR-cyclase and/or
cADPR. The compounds and composition of the invention can be used to
selective and specific inhibition of ADPR-cyclase. An activation of
ADPR-cyclase leads to an increase of intracellular calcium levels, which
are related on blood pressure overload and glucose homeostasis. Thus, the
compounds of the invention may further be used to treatment and
prevention of hypertension, diabetes, and diabetic nephropathy.
Accordingly, the compounds and compositions of the invention can be used
for treatment and prevention of cardiovascular disease and related
disease states, particularly, hypertension or diabetes related disorders,
such as, hypertensive cardiac hypertrophy, diabetic nephropathy, and the
like.

Advantageous Effects

[0026] The compounds and compositions of the invention can be used for
treatment and prevention of cardiovascular disease and related disease
states, particularly, hypertension or diabetes related disorders, such
as, hypertensive cardiac hypertrophy, diabetic nephropathy, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGS. 1A and 1B show the inhibition of rat kidney ADPR-cyclase that
was incubated with 200 mM NGD.sup.+ or e-NAD.sup.+ in the presence of
various concentrations of 4,4'-dihydrioxyazobenzene (DHAB) at 37°
C. for 10 min and effects of DHAB on human CD38, rat brain, heart,
spleen, and kidney ADPR-cyclase. FIGS. 1A and 1B are referred to as FIG.
1 herein.

[0028] FIGS. 2A and 2B show the inhibition effects of 4,4'-DHAB on
angiotensin II (Ang II) induced [Ca2+]i increase and cADPR
production in murine mesangial cells (MMCs). FIGS. 2A and 2B are referred
to as FIG. 2 herein.

[0029] FIGS. 3A and 3B show the inhibition effects of 2,2'-DHAB on
angiotensin II (Ang II) induced intracellular Ca2+
([Ca2+]i) increase and cADPR production in rat cardiomyocytes.
FIGS. 3A and 3B are referred to as FIG. 3 herein.

[0030] FIGS. 4A and 4B show the inhibition effects of 4,4'-DHAB or
2,2'-DHAB on OKT3, which is a ligand for CD3/TCR, induced
[Ca2+]i increase and cADPR production in Jurkat T cells. FIGS.
4A and 4B are referred to as FIG. 4 herein.

Assays for ADPR-Cyclase or cADPR-Hydrolase and Inhibition Effects of
4,4-DHAB in a Dose Dependent Manner

[0032] Specific ADPR-cyclase activity was determined by measuring cyclic
GDP-ribose (cGDPR) or etheno-ADP-ribose (e-cADPR) fluorometrically using
NGD.sup.+ or e-NAD.sup.+ as a substrate [Greaff et al., Biochem. J.
(2002)361:379-84]. Samples were incubated in the presence of 200 mM
NGD.sup.+ or e-NAD.sup.+ with and without an appropriate agent in an
assay buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, and 0.1% Triton X-100) in
a 50 ml-final volume. The reaction mixture was incubated at 37° C.
for 10 min. The reaction was stopped by adding 50 ml trichloroacetic acid
(10%). The samples were centrifuged at 21,000 g for 10 min and the
supernatant (80 ml) was diluted with 920 ml of 100 mM sodium phosphate
buffer (pH 7.2). Fluorescence of cGDPR or e-ADPR in the solution was
determined at excitation/emission wavelengths of 297/410 nm (HITACHI
F-2000 fluorescence spectrophotometer). cADPR-hydrolase activity was
determined by incubating cADPR with ADPR-cyclase or CD38 at 37° C.
for 20 min. Hydrolysis of cADPR was analyzed by high performance liquid
chromatography as described (White et al., 2000). The results provided in
FIG. 1 that 4,4'-DHAB was able to inhibit generation of cGDPR and e-ADPR
from NGD.sup.+ and e-NAD.sup.+, respectively, by the kidney ADPR-cyclase
in a concentration-dependent manner. These results suggest that the
compound may bind to the active site of the enzyme. Half maximal
inhibition (IC50) of the enzyme activity was approximately 100 mM.
CD38 and ADPR-cyclases purified partially from rat brain, heart, and
spleen tissues were insensitive to 4,4'-DHAB at 200 mM.

[0035] IC50 of the compounds for cADPR production and the later
sustained Ca2+ signal induced by 150 nM Ang II was determined after
incubation for 90 s ([c ADPR]i) and 300 s ([Ca2+]i).

[0036] Ang II generates of long-lasting increase of [Ca2+]i, a
burst Ca2+ rise followed by a sustained Ca2+ rise that was
gradually decreased (FIG. 2). The sustained Ca2+ signal, but not the
initial burst Ca2+ rise, was blocked by pretreatment with 4,4'-DHAB
as a possible candidate inhibitor of ADPR-cyclase. IC50 was
approximately 2.5 nM (See the above Table 1).

[0038] Changes in [Ca2+]i in Jurkat T cells were determined as
described above examples 1. The results provided in FIG. 4. Treatment of
Jurkat T cells with OKT3 (5 mg/ml), which is a ligand for CD3/TCR, showed
a typical biphasic increase of [Ca2+]i an initial peak rise
followed by a sustained rise. Pretreatment with 4,4'-DHAB as well as
2,2'-DHAB did not show any effects on OKT3-mediated Ca2+ rise even
at 10 mM (FIG. 4). These results obtained from in vitro study indicated
that DHAB analogues are specific for the kidney or heart ADPR-cyclase.

[0039] [cADPR]i was measured using a cyclic enzymatic assay as
described previously [Graeff et al., Biochem. J. (2002)361:379-84].
Aplysia californica ADPR-cyclase was purified from sea urchin egg
according to the method described [Lee et al., Cell Regul.
(1991)2:203-9]. Briefly, MMCs were treated with 0.3 ml of 0.6 M
perchloric acid under sonication after Ang II treatment. Precipitates
were removed by centrifugation at 20,000×g for 10 min. Perchloric
acid was removed by mixing the aqueous sample with a solution containing
3 volumes of 1,1,2-trichlorotrifluoroethane to 1 volume of
tri-n-octlyamine. After centrifugation for 10 min at 1500×g, the
aqueous layer was collected and neutralized with 20 mM sodium phosphate
(pH 8). To remove all contaminating nucleotides, the samples were
incubated with the following hydrolytic enzymes overnight at 37°
C.: 0.44 unit/ml nucleotide pyrophosphatase, 12.5 units/ml alkaline
phosphatase, 0.0625 unit/ml NADase, and 2.5 mM MgCl2 in 20 mM sodium
phosphate buffer (pH 8.0). Enzymes were removed by filtration using
Centricon-3 filters. To convert cADPR to NAD.sup.+, the samples (0.1
ml/tube) were incubated with 50 ml of a cycling reagent containing 0.3
mg/ml Aplysia ADPR-cyclase, 30 mM nicotinamide, and 100 mM sodium
phosphate (pH 8) at room temperature for 30 min. The samples were further
incubated with the cycling reagent (0.1 ml) containing 2% ethanol, 100
mg/ml alcohol dehydrogenase, 20 mM resazurin, 10 mg/ml diaphorase, 10 mM
riboflavin 5'-phosphate, 10 mM nicotinamide, 0.1 mg/ml BSA, and 100 mM
sodium phosphate (pH 8.0) for 2 h at room temperature. An increase in the
resorufin fluorescence was measured at 544 nm excitation and 590 nm
emission using a fluorescence plate reader (Molecular Devices Corp.,
Spectra-Max GEMINI). Various known concentrations of cADPR were also
included in the cycling reaction to generate a standard curve. The
results provided in FIG. 2 and Table 1. These data show that 4,4'-DHAB
inhibits cADPR production stimulated by Ang II in MMC at=5 nM and is far
more potent in cell-based system than in vitro. Production of cADPR as
well as later sustained Ca2+ signal in response to Ang II was
inhibited with different efficacy by these small molecules (Table 1). As
expected, among these small molecules, DAHB showed the strongest
inhibitory potency. The order of IC50 was
4,4'-DAHB>resveratrol>azobenzene=piceatannol>2,2'-DAHB. These
observations suggest that the biphenyl moiety but not the azo bond and
position of hydroxyl group affect the binding of the inhibitor to the
enzyme.

Example 6

Measurement of Ang II-Induced [cADPR]i in Cardiomyocytes

[0040] Measurement of [cADPR]i in cardiomyocytes were determined as
described above examples 5. The results provided in FIG. 3. The
production of cADPR was increased approximately 5 times more than control
by Ang II in cardiomyocytes. Pretreatment with 2,2-DHAB inhibited the Ang
II-mediated [cADPR]i in cardiomyocytes.

Example 7

Measurement of OKT3-Induced [cADPR]i in Jurkat T Cells

[0041] Measurement of [cADPR]i in Jurkat T cells were determined as
described above examples 5. The results provided in FIG. 4. The
production of cADPR was increased approximately 2 times more than control
by OKT3 in Jurkat T cells. Pretreatment with 2,2'-DHAB or 4,4'-DHAB
didn't inhibited the OKT3-mediated [cADPR]i in Jurkat T cells. These
results indicate that DRAB analogues selectively inhibited kidney or
heart ADPR-cyclase.

[0042] Renovascular hypertension was produced by 2KlC Sprague-Dawley male
rats (7-9 weeks old) weighing 200 to 220-g were anesthetized with
ketamine (100 mg/kg, intra-peritoneally) and rumpen (5 mg/kg,
intraperitoneally). The left kidney was exposed through the median
abdominal incision, and the renal artery was separated from the renal
vein with caution. Then, a silver clip with 0.15 mm slit was placed
around the renal artery. The sham procedure was performed, including the
entire surgery with the exception of arterial clipping. To examine the
effect of 2,2'-DHAB in the 2KlC model, we administered it at days 7 after
the surgery for 7 weeks. 2,2'-DHAB was injected intraperitoneally with
58% DMSO plus saline at a dose of 1.5 μl/g body weight (428 μg/200
g/d). Sham group and control 2KlC received DMSO plus saline treatment.
The dose of injected DMSO had no side effect on blood pressure (BP) or
any other parameters. Systolic blood pressure was measured by a method
using tail plethysmography in conscious rats once a week, from the day
prior to surgery until the day of sacrifice. The results provided in FIG.
5 in which hypertensive control groups showed elevation of systolic blood
pressure after 2 weeks but 2,2'-DHAB treated hypertensive groups
prevented the elevation of blood pressure.

[0043] Male mice, weighing 20 25 g, were made diabetic by a single
intravenous injection of STZ (65 mg/kg body weight) in 0.05 M citrate
buffer (pH 4.8). At the same day, the control mice were injected with the
citrate buffer. After 2 days, induction of diabetes was confirmed by tail
blood glucose level measurement by using the LifeScan One Touch
glucometer (Johnson & Johnson). The diabetic mice (>16 mM blood
glucose) were randomly divided into two groups; 6 mice per group treated
with vehicle (0.1% DMSO in saline, 100 μl) or DHAB (45 μg/kg body
weight in 0.1% DMSO in saline, 100 μl) and resveratrol (45 μg/kg
body weight in 0.1% DMSO in saline, 100 μl) administered by
intraperitoneal injection once a day for 6 weeks. The control mice were 6
mice per group treated with the vehicle. On day 39, the mice were
detained in individual metabolic cages for 24 h for urine collection. On
day 42, the mice were anesthetized with diethyl ether, and blood samples
were taken from the abdominal aorta. Bilateral kidneys were rapidly
removed and weighed. Urine was gravimetrically collected, and urinary
albumin concentrations were determined with an enzyme-linked
immunosorbent assay using a murine microalbuminuria kit (Albuwell;
Exocell, Philadelphia, Pa.). Urine and serum creatinine levels were
measured using the QuantiChrom Creatinine Assay Kit (BioAssay Systems,
Hayward, Calif.), following the manufacturer's protocol. The results
provided in Table 2.

[0045] As shown in Table 2, administration of STZ to mice significantly
enhanced the levels of blood glucose, urinary albumin, and creatinine
clearance (CCr) compared to those in vehicle control mice. These data
show the characteristics of diabetic renal dysfunction and establishment
of diabetes mice. Moreover, treatment of diabetes mice with DHAB or
resveratrol significantly recovered the urinary albumin, and CCr, but not
the level of blood glucose, suggesting that bisphenyl analogues attenuate
the progression of diabetic nephropathy, but not diabetes.

[0046] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and practice
of the invention disclosed herein. It is intended that the specification
and examples be indicated by the following claims.